The objective of this project is to identify structural features that determine ion selectivity and regulation mechanisms of heavy (transition) metal transport ATPases (CPx-ATPases). These P-type ATPases transport metals (Cu+, Ag+, Zn2+, Co2+, Cd2+, Pb2+) across biomembranes and against their concentration gradients. These are key enzymes in the mechanisms of heavy metal absorption, distribution, and bioaccumulation. Members of this protein family are responsible for Wilson and Menkes' disease in humans. Simpler CPx-ATPases are found in prokaryotes and they appear as suitable models for this family of transporters. A transmembrane metal binding "CPx" site ([C/S]P[C/H/S]) and one to six cytoplasmic metal binding repeats in the N-terminus of the protein characterize the CPx-ATPases. While putative transport mechanism has been proposed for CPx-ATPases based on their structural homology with other P-type ATPases (Na,K-ATPase, Ca-ATPase), their transport stoichiometry, regulatory mechanisms, and determinants of metal ion specificity are not known. In this proposal, using prokaryote enzymes with different predicted ion selectivity, we plan to test whether the signature CPx sequence and/or conserved sequences in the seventh and eighth transmembrane segments of the protein determine ion specificity. In addition, we will test the putative role of the cytoplasmic metal binding site in ion selectivity and as a regulatory domain. Results from these studies will help to establish the mechanism of heavy metal binding to carrier proteins and increase our understanding of heavy metal transport.